1. Field of the Invention
The present invention relates to a lyophilized composition of recombinant factor VIII used as a therapeutic preparation of hemophilia A, and more particularly, to a lyophilized composition recombinant factor VIII without albumin using an amino acid as a stabilizer for stabilizing the recombinant factor that is unstable in activity during lyophilization, the amino acid being free from a risk of viral infection.
2. Description of the Related Art
Hemophilia is one of inherited coagulation disorders and is known to be caused due to a shortage of biologically active coagulation factors, which are normally present in the blood human blood plasma.
Since hemophilia is an inherited sex-linked recessive bleeding disease, females are very rarely affected even if the females have hemophilia genes, affecting mainly the male population. The hemophilia is classified into hemophilia A, hemophilia B, hemophilia AB and hemophilia C according to the type of deficient blood clotting factor.
Hemophilia A affects 1-2 individuals per 20,000 males due to deficiency or absence of human in vivo blood coagulating factor VIII (antihemofilic factor). Hemophilia B occurs in about 1 of 100,000 males due to deficiency of human blood coagulation factor IX (Christmas factor). Hemophilia AB reflects a deficiency of human recombinant blood coagulating factors VIII and IX, and hemophilia C reflects a deficiency of human recombinant coagulating factor XI.
Therapeutic agents for the treatment of hemophilia A have been produced for the last three decades by isolating the factor VIII from human blood plasma, purifying and concentrating the same. Such therapeutic agents have allowed many hemophiliacs to lead a normal life.
However, the human blood derived coagulating factor VIII, which is isolated from human blood plasma to be used as the conventional therapeutic agents for the treatment of hemophilia, encountered certain disadvantages, including susceptibility to infection with blood-mediated viruses such as hepatitis, human immunodeficiency virus (HIV) or TT virus. To overcome such problems, as reported in J. Gitschier et al., Nature 312, 330-37, 1984 and EP 160 457, preparation of recombinant human coagulating factor VIII generally involves isolating the same from recombinant animal cell culture products using recombinant DNA technology and purifying the isolated coagulating factors.
The structure and biochemical reaction scheme of recombinant factor VIII products are described in Kaufman Tibtech, Vol 9, 1991 and Hematology, 63, 155-65, 1991.
Human factor VIII concentrates isolated from human blood plasma contain several fragmented factor VIII forms of stable activity (see Andersson et al., Proc. Natl. Acad. Sci. USA, Vol 83, 2979-83, May 1986). The smallest active form of factor VIII has a molecular weight of about 170 kDa and is composed of two chains of 90 kDa and 80 kDa fragments linked to each other by metal ion crosslinkage (see EP 197 901). Kabi Pharmacia developed recombinant factor VIII products of 170 kDa with a B-domain deleted from human blood derived coagulating factor VIII. The severed recombinant factor, which is termed r-VIII SQ, and is produced by recombinant Chinese Hamster ovary (CHO) cell line from serum-free media by animal cell cultivation. The structure and biochemical reaction scheme of r-VIII SQ are described in WO 91/09122.
Recombinant factor proteins are produced in a lyophilized form to be commercially distributed for better preservation, storage and handling. For being administered to a patient, the lyophilized proteins are reconstituted in an appropriate solvent. During lyophilization, however, the recombinant factor proteins may experience a considerable reduction in the activity while undergoing purification, sterilization, lyophilization, packaging and reconstitution for injection. Also, dry cake appearance is undesirably poor. Thus, a conventional attempt was made to achieve stabilization of recombinant factor using human blood derived albumin as a stabilizer in lyophilization. The human blood derived albumin serves as a general stabilizer in purification and sterilization as well as lyophilization (see Wang et al., J. of Parenteral Sci. and Tech. Vol 42, Number 2S, Supplement. 1988). Also, the human blood derived albumin is a good cake-forming agent in formulations for lyophilization. The use of albumin in stabilizing recombinant factor VIII is widely known in the art, and albumin is also employed to some products of highly purified recombinant factor VIII that are currently commercially available. However, since the human blood derived albumin is isolated from human blood plasma, it is vulnerable to infection with blood-derived viruses such as hepatitis, HIV, or TT virus. Therefore, the human blood derived albumin cannot be suitably used for hemophilia therapeutic agents produced by recombinant DNA technology. Further, when a physicochemical test is performed on final products, excess albumin relative to a small concentration of main pharmaceutically effective components may cause interference, making accurate quality control difficult.
Thus, it would be desirable to provide a pharmaceutical preparation of recombinant factor without albumin, which is stable in a solution during lyophilization or as a solution resulting after lyophilization and reconstitution.
To stabilize recombinant factors, various attempts have been proposed. For example, U.S. Pat. No. 5,763,401 (EP 818 204, Bayer) discloses a method of stabilizing recombinant factor using 15 to 60 mM of sucrose as a stabilizer.
U.S. Pat. No. 5,733,873 (EP 627 924, Pharmacia & Upjohn) discloses a stabilization method of a recombinant factor using 0.01 to 1 mg/ml of a non-ionic surfactant (polysorbate 20 or 80) as a stabilizer.
U.S. Pat. No. 4,877,608 (EP 315 968, Rhone-Poulenc Rorer) discloses a stabilization method of a recombinant factor using low concentration ions, that is, 0.5 to 15 mM of NaCl, KCl and 0.01 to 10 mM of lysine hydrochloride, to which sugar such as maltose, sucrose or mannitol may be further added.
U.S. Pat. No. 5,605,884 (EP 314 095, Rhone-Poulenc Rorer) discloses a stabilization method of a recombinant factor using high concentration ions, that is, 0.35 to 1.2 M NaCl, and 1.5 to 40 mM KCl, to which saccharides such as maltose, sucrose or mannitol may also be further added.
International Patent Application WO 96/22107 (Quadrant Holings Cambrige Limited) discloses use of trehalose as a stabilizer.
International Patent Application WO 89/09614 (Genentech) describes a formulation having stabilized human growth hormone including glycine, mannitol and a buffer. In a preferred embodiment of the formulation, a non-ionic surfactant, such as polysorbate 80, is added thereto. The non-ionic surfactant is added for reduced aggregation and denaturation. The prepared formulation exhibits increased stability during lyophilization and after reconstitution.
EP 268 110 (Cetus) describes a solution comprising a particular protein dissolved in an inactive carrier medium containing a non-ionic polymer clarificant as a solvent/stabilizer, for example, interleukin-2. Preferred examples of the clarificant include octylphenoxy polyethoxy ethanol compound, polyethylene glycol monostearate compound and polyethylene sorbitan fatty acid ester.
U.S. Pat. No. 4,783,441 (Hoechst) describes an aqueous solution containing proteins, for example, insulin and a pulmonary surfactant.
U.S. Pat. No. 165,370 (Coval) discloses a gamma globulin solution and a preparation method thereof. The disclosed solution contains polyethylene glycol (PEG). The solution may further include a non-ionic surfactant.
EP 77 870 (Japan Green Cross) discloses adding amino acids, monosaccharides, oligosaccharides, sugar alcohol or hydrocarbon carboxyl acid to a solution containing a recombinant factor in order to improve stability of the solution. EP 117064 describes adding sugar alcohol or disaccharide to an aqueous solution of a recombinant factor in order to increase stability during annealing.
International Patent Application WO 91/10439 (Octopharma) discloses a stable injectable solution of factor VIII or factor IX containing disaccharide, preferably saccharose, and one or more kinds of amino acids.